Fluoresence Polarization (FP) is a measure of the time-average rotational motion of fluorescent molecules. It has been known since the 1920's and has been used in both research and clinical applications for sensitive determination of molecular volume and microviscosity. The FP technique relies upon changes in the rotational properties of molecules in solution. That is, molecules in solution tend to "tumble" about their various axes. Larger molecules (e.g., those with greater volume or molecular weight) tumble more slowly and along fewer axes than smaller molecules. There is therefore less movement between excitation and emission, causing the emitted light to exhibit a relatively higher degree of polarization. Conversely, fluorescence emissions from smaller fluorescent molecules, which exhibit more tumbling between excitation and emission, are more multiplanar (less polarized). When a smaller fluorescent molecule takes a larger or more rigid conformation its tumbling decreases and the emitted fluorescence becomes relatively more polarized. This change in the degree of polarization of emitted fluorescence can be measured and used as an indicator of increased size and/or rigidity of the fluorescent molecule.
In fluorescence polarization techniques, the fluorescent molecule is first excited by polarized light. The polarization of the emission is measured by measuring the relative intensities of emission (i) parallel to the plane of polarized excitation light and (ii) perpendicular to the plane of polarized excitation light. A change in the rate of tumbling due to a change in size and/or rigidity is accompanied by a change in the relationship between the plane of excitation light and the plane of emitted fluorescence, i.e., a change in fluorescence polarization. Such changes can occur, for example, when a single stranded oligonucleotide probe becomes double stranded or when a nucleic acid binding protein binds to an oligonucleotide. Fluorescence anisotropy is closely related to FP. This technique also measures changes in the tumbling rates of molecules but is calculated using a different equation. It is to be understood that polarization and anisotropy are interchangeable techniques for use in the present invention. The term fluorescence polarization is generally used herein but should be understood to include fluorescence anisotropy methods. In steady state measurements of polarization and anisotropy, these values are calculated according to the following equations: ##EQU1## where Ipa is the intensity of fluorescence emission parallel to the plane of polarized excitation light and Ipe is the intensity of fluorescence emission perpendicular to the plane of polarized excitation light.
As FP is homogenous, this technique is ideal for studying molecular interactions in solution without interference by physical manipulation. Fluorescence polarization is therefore a convenient method for monitoring conversion of single-stranded fluorescently labelled DNA to double-stranded form by hybridization (Murakami, et al. 1991. Nucl. Acids Res. 19, 4097-4102). The ability of FP to differentiate between single and double-stranded nucleic acid conformations without physical separation of the two forms has made this technology an attractive alternative for monitoring probe hybridization in diagnostic formats. European Patent Publication No. 0 382 433 describes fluorescence polarization detection of amplified target sequences by hybridization of a fluorescent probe to the amplicons or by incorporation of a fluorescent label into the amplification products by target-specific extension of a fluorescently-labeled amplification primer. PCT Patent Publication No. WO 92/18650 describes similar methods for detecting amplified RNA or DNA target sequences by the increase in fluorescence polarization associated with hybridization of a fluorescent probe.
Fluorescence polarization may be monitored in any of three different states: steady state, transient state, or dynamic state. In transient state FP, the excitation light source is flashed on the sample and polarization of the emitted light is monitored by turning on the photomultiplier tube after the excitation light source is turned off. This reduces interference from light scatter, as fluorescence lasts longer than light scatter, but some fluorescence intensity is lost. In steady state FP, excitation light and emission monitoring are continuous (i.e., the excitation source and photomultiplier tube are on continuously). This results in measurement of an average tumbling time over the monitoring period and includes the effects of scattered light. Dynamic FP may be monitored in either the time- or frequency-domain. Dynamic fluorescence techniques involve determining the lifetime of the fluorescent molecule in nanoseconds. The theory of dynamic fluorescence monitoring is described in "Principles of Fluorescence Spectroscopy" (Lakowicz, Plenum Press, 1983). Whereas steady state FP provides an average or "snapshot" of the fluorescence phenomena, dynamic FP allows one to observe the individual contributions of the fluorescent components in the system being studied. Use of these three fluorescence techniques is described by Kumke, et al. (1995. Anal. Chem. 67, 3945-3951), Devlin, et al. (1993. Clin. Chem. 39, 1939-1943), and Walker, et al. (1995. Clin. Chem. citation omitted).
Analysis of nucleic acids, and in particular detection of specific nucleic acid target sequences provides an extremely sensitive tool for diagnosis and identification of biological materials. Typically, nucleic acid target sequences are detected by specific hybridization to a labeled oligonucleotide probe. Several probe hybridization methods for detecting nucleic acid target sequences are known in the art (e.g., dot blots, Southern blots, Northern blots), but these are somewhat insensitive and are generally only applicable to samples containing relatively large amounts of the target sequence to be detected. Nucleic acid amplification techniques have greatly improved the sensitivity of target sequence detection by providing methods for specifically increasing the amount of target sequence prior to detection. Nucleic acid amplification methods can be grouped according to the temperature requirements of the procedure. The polymerase chain reaction (PCR; R. K. Saiki, et al. 1985. Science 230, 1350-1354), ligase chain reaction (LCR; D. Y. Wu, et al. 1989. Genomics 4, 560-569; K. Barringer, et al. 1990. Gene 89, 117-122; F. Barany. 1991. Proc. Natl. Acad Sci. USA 88, 189-193) and transcription-based amplification (D. Y. Kwoh, et al. 1989. Proc. Natl. Acad. Sci. USA 86, 1173-1177) require temperature cycling. In contrast, methods such as Strand Displacement Amplification (SDA; G. T. Walker, et al. 1992. Proc. Natl. Acad. Sci. USA 89, 392-396 and G. T. Walker, et al. 1992. Nuc. Acids. Res. 20, 1691-1696, and U.S. Pat. No. 5,455,166), self-sustained sequence replication (3SR; J. C. Guatelli, et al. 1990. Proc. Natl. Acad Sci. USA 87, 1874-1878), Nucleic Acid Sequence Based Amplification (U.S. Pat. No. 5,409,818), restriction amplification (U.S. Pat. No. 5,102,784) and the Q.beta. replicase system (P. M. Lizardi, et al. 1988. BioTechnology 6, 1197-1202) are isothermal reactions. Isothermal amplifications are conducted at essentially constant temperature, in contrast to the cycling between high and low temperatures characteristic of amplification reactions such as the PCR. The SDA reaction originally reported in the publications cited above ("conventional SDA") is typically conducted at a temperature between about 35.degree. C. and 42.degree. C., and is capable of 10.sup.8 -fold amplification of a target sequence in about 2 hours. Recently, SDA has been adapted for higher reaction temperatures (about 45.degree.-65.degree. C.--"thermophilic SDA" or "tSDA"). tSDA is capable of producing 10.sup.9 -10.sup.10 fold amplification in about 15-30 min. at about 50.degree.-60.degree. C. In addition to increased reaction speed, there is a significant reduction in non-specific background amplification in tSDA as compared to conventional SDA.
Either unamplified or amplified target sequences may be detected by hybridization of a labeled oligonucleotide probe. This often requires separation of free and hybridized probe before the signal can be measured. However, monitoring changes in FP allows differentiation of free and hybridized probe without physical separation, thereby reducing operating steps and procedural complexity. As an alternative to probe hybridization, target amplification may be detected by generating double-stranded secondary amplification products from a single-stranded signal primer in a target amplification-dependent manner during the amplification reaction. Generation of secondary amplification products during target amplification is described and illustrated in published European Patent Application Nos. 0 678 582 and 0 678 581. In the process, a single-stranded oligonucleotide signal primer comprising a detectable label is converted to double-stranded form in a target amplification-dependent manner. Conversion of the signal primer occurs concurrently with the amplification reaction and may be detected as a change in FP when the label is fluorescent. The increase in FP associated with conversion of the signal primer to double-stranded form as a result of target amplification is approximately 20 mP using fluorescein or La Jolla Blue as the fluorescent label. When amplification is conducted at lower temperatures (e.g., about 35.degree.-45.degree. C.), the change in FP can be enhanced (e.g., to about 133-185 mP) by binding a double-stranded DNA binding protein to its specific binding sequence incorporated into the signal primer. In this system, enhancement is amplification-specific because protein binding can occur only when the binding sequence in the signal primer becomes double-stranded as a result of target amplification. At temperatures less than about 45.degree. C., where the duplex is entirely double-stranded, enhancement of polarization is probably primarily the result of the DNA binding protein further slowing the tumbling time of the molecule.
The specificity of probe hybridization and/or amplification is increased at higher temperatures (e.g., 45.degree.-75.degree. C.). It is therefore desirable to combine the advantages of FP for detecting nucleic acid target sequences with elevated reaction temperatures. However, increased temperature was expected to be incompatible with FP detection. Many fluorescent labels are not stable at higher temperatures. In addition, higher temperatures promote "breathing" of the duplex and "fraying" of the ends, leading to increased single-strandedness. This increased single-strandedness near the fluorescent label, particularly at the end of the duplex, could significantly decreases the magnitude of the change in FP for the double-stranded form and potentially eliminate it at temperatures which are optimized for hybridization specificity. These concerns were supported by preliminary experiments evaluating the change in FP upon hybridization at 55.degree. C. At this temperature there was no difference in polarization between the single-stranded and double-stranded forms of oligonucleotides. Further, FP is sensitive to sample viscosity, which is altered at higher temperatures. The effects of altered sample viscosity on the ability to use changes in FP for detection of nucleic acid target sequences at increased reaction temperatures were therefore uncertain.